We studied the distribution pattern of the radiotracer 14C in ryegrass at three different time intervals: 6 h, 2 d and 11 d after start of assimilation of 14CO2 via the shoots of the plants. The 14C distribution pattern was visualized using phosphor imaging of leaves and roots. The main focus of this study was on the 14C distribution in roots.
1.2.1 Soil properties and plant growth conditions
The experiments were conducted with Lolium perenne grown on a fine loamy gleyic Cambisol. The soil samples were taken from the Ah horizon (top 10 cm) of a long-term pasture in Allgäu (S Germany). Basic characteristics of the soil are shown in Table II.1/1.
Table II.1/1: Basic characteristics of the soil sampled from the Ah horizon of a fine loamy gleyic Cambisol from a pasture in the Allgäu (S Germany) (FC, field capacity (pF=1.8); AWC, available water capacity (pF 1.8-4.2)) (Kleber, 1997).
Parameter Value
pH (CaCl2) 5.2
Corg % 4.7
Nt % 0.46
C/N 10.0
Clay (<2 µm) % 28.4 Silt (2-<63 µm) % 47.1 Sand (63-2000 µm) % 24.5
FC % 50.0
AWC % 23.0
CaCO3 % 0.0
The wet soil samples were air dried, homogenized, and passed through a 2 mm sieve to separate large roots and stones. An amount of 1.6 kg of dried soil with a final density of 1.2 g cm-³ was filled into each pot (height 10 cm, inner diameter 14 cm). One pre-vernalized seedling of ryegrass was grown per pot. The plants were grown at tempera-tures of 26°C-28°C (day) and at 22°C-23°C (night) with a day length of 14 h and light
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intensity of ≈ 800 µmol m² s . The soil water content of each pot was measured gravi-metrically and was adjusted daily to ≈ 60% of the available field capacity.
1.2.2 Labeling of plants in a 14CO2 atmosphere
For 14C labeling a perspex chamber previously described by Kuzyakov et al. (1999) was used. The airtight chamber consisted of two compartments. The lower compartment (height 200 mm, inner diameter 138 mm) contained the soil, and the upper compartment (height 300 mm, inner diameter 138 mm) was used for the tracer application to the leaves. Both compartments were separated from each other by a perspex lid with drilled holes (inner diameter 8 mm) for the plants. The day before labeling, the holes were sealed at the base using silicone paste (NG 3170, Thauer & Co., Dresden) (Gregory et al., 1991; Swinnen et al., 1995 a). The seals were tested for air leaks. All plants were labeled simultaneously. 381 kBq of 14C as Na214
CO3 solution were put in a 2 cm³ Ep-pendorf micro test tube placed in the upper compartment of the chamber. Then the chamber was closed and 1 ml of 5 M H2SO4 was added to the solution through a pipe.
Assimilation took place within 2 h after the 14CO2 pulsing, but most of the 14CO2 was assimilated within the first 30 min. After labeling, the CO2 from the upper compartment was trapped to remove the remaining non-assimilated 14CO2. Finally, the top of the chamber was removed and the plants were grown under normal conditions.
After opening the labeling chamber, the CO2 evolving from the lower compartment was trapped in a 20 mL solution of 0.5 M NaOH by continuous pumping (100 cm³ min-1) with a membrane pump. This removes the 14CO2 respired by roots and microorganisms and avoids possible re-uptake of 14C from the soil solution by roots.
The plants were harvested at three different times after start of labeling: 6 h, 2 d and 11 d. This was done by cutting the plants at the base and opening the bottom compartment of the chamber. Finally, the soil was pulled out. Roots were carefully separated from the soil by handpicking. All picked roots were gently washed in 400 mL of deionized water to remove the soil adhering to the roots. The leaf material and the roots were distributed on a white paper, prepared as a herbarium and dried at 60°C.
46 1.2.3 Tracer detection by phosphor imaging
The distribution pattern of the 14C within leaves and roots was determined by Cyclone-Plus Storage Phosphor System (Perkin Elmer, Germany). Each herbarium (6 h, 2 d, 11 d) with roots or shoots was exposed to a sensitive imaging plate in the dark for 1 or 3 weeks. The plate was then scanned by CyclonePlus (Perkin Elmer, Germany) and digi-talized by OptiQuant software (Perkin Elmer, Germany). We used two approaches to demonstrate the distribution pattern of assimilated 14C within the roots: 1) evaluation of the evenness of the 14C distribution within the roots and identification of 14C hotspots and 2) visualization of the longitudinal allocation in individual roots.
1.2.3.1 Evenness of the 14C distribution within the roots and identification of 14C hotspots
To verify the visual findings of the image-plate pictures, the evenness of the 14C distri-bution within the roots was calculated and hotspots were identified by applying a grid to each image (241 columns, 122 rows, square width 1 mm, square length 1 mm, center to center spacing: columns 1 mm, rows 1 mm). The 14C activities of the single squares were added up and set as the total activity of the grid. The activity per square expressed as digital light units (DLU) per mm² was then put in reference to the total activity. The resulting relative activities were categorized into 39 size ranges with the statistical package Statistica7 for Windows. The smallest range with a relative activity of ≤ 0.006 (> 82% of the image area) was set up as background and excluded from the evaluation.
1.2.3.2 Longitudinal 14C allocation in individual roots
Within each image, 10 roots were selected and squares (1 mm x 1 mm) were applied to the individual roots in longitudinal direction up to 19 mm. The squares along each root were numbered starting at the root tip. Subsequently, the mean out of all squares of #1,
#2, etc. per image was calculated and the data were normalized with reference to the square with the maximum DLU value out of all images. In this approach we used another reference to normalize the data because, despite the same root length, the area around the roots differs and thus cannot be normalized as described under section 1.2.3.1.
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